In the past decade, a great deal of progress has been made in the field of targeted genome engineering. Technologies such as designer zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and homing meganucleases have made site-specific genome modifications a reality in many different model organisms ranging from zebrafish to mammalian cells. Based on the results to date, however, genome editing tools that are efficient, flexible, and cost-effective have remained elusive to the general research community. The recent discovery of the type II prokaryotic CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, originally discovered in the bacterium Streptococcus pyogenes as a mechanism to defend against viruses and foreign DNA, has provided yet another tool for targeted genome engineering, this time taking advantage of a system that uses small RNAs as guides to cleave DNA in a sequence-specific manner. With its ease in designing guide sequences to target specific sequences (unlike ZFNs and TALENs where construct assembly can be laborious and time-consuming), as well as its targeting efficiency, this system has the potential to be a disruptive technology in the field of genome-engineering.
The CRISPR/CRISPR-associated (Cas) system involves 1) retention of foreign genetic material, called “spacers”, in clustered arrays in the host genome, 2) expression of short guiding RNAs (crRNAs) from the spacers, 3) binding of the crRNAs to specific portions of the foreign DNA called protospacers and 4) degradation of protospacers by CRISPR-associated nucleases (Cas). A well-characterized Type II CRISPR system has been previously described in the bacterium Streptococcus pyogenes, where four genes (Cas9, Cas1, Cas2, Csn1) and two non-coding small RNAs (pre-crRNA and tracrRNA) act in concert to target and degrade foreign DNA in a sequence-specific manner [Jinek et. al. 2012]. The specificity of binding to the foreign DNA is controlled by the non-repetitive spacer elements in the pre-crRNA, which upon transcription along with the tracrRNA, directs the Cas9 nuclease to the protospacer:crRNA heteroduplex and induces double-strand breakage (DSB) formation. Additionally, the Cas9 nuclease cuts the DNA only if a specific sequence known as protospacer adjacent motif (PAM) is present immediately downstream of the protospacer sequence, whose canonical sequence in S. pyogenes is 5′-NRG-3′, where N refers to any nucleotide and R refers to A or G.
There are a myriad of examples in the art disclosing variations on themes of multiple promoters used in the same vector.
One potential problem with vectors designed to accommodate expression of two inserts under control of separate promoters is that insertion of both inserts may require sequential cloning and isolation steps as well as using of different selection markers for such inserts. The additional effort becomes even more of a concern if it is desired to introduce libraries of inserts rather than single inserts under control of each promoter and only select combinations of the inserts from the respective libraries are compatible with one another.
Therefore, there remains a need for new classes of expression vectors and related methods suitable for expression of multiple small guide RNA molecules or other nucleic acids intended to be expressed in a pair-wise fashion.